Ovarian cancer cells direct monocyte differentiation through a non-canonical pathway

Alyssa Walker,Kaitlin C Fogg,Andrew E Miller,Will Flanigan,Andrea O’Shea,Christina Kendziorski,Pamela K Kreeger,Ying Li

doi:10.1186/s12885-020-07513-w

Abstract

BackgroundAlternatively-activated macrophages (AAMs), an anti-inflammatory macrophage subpopulation, have been implicated in the progression of high grade serous ovarian carcinoma (HGSOC). Increased levels of AAMs are correlated with poor HGSOC survival rates, and AAMs increase the attachment and spread of HGSOC cells in vitro. However, the mechanism by which monocytes in the HGSOC tumor microenvironment are differentiated and polarized to AAMs remains unknown.MethodsUsing an in vitro co-culture device, we cultured naïve, primary human monocytes with a panel of five HGSOC cell lines over the course of 7 days. An empirical Bayesian statistical method, EBSeq, was used to couple RNA-seq with observed monocyte-derived cell phenotype to explore which HGSOC-derived soluble factors supported differentiation to CD68+ macrophages and subsequent polarization towards CD163+ AAMs. Pathways of interest were interrogated using small molecule inhibitors, neutralizing antibodies, and CRISPR knockout cell lines.ResultsHGSOC cell lines displayed a wide range of abilities to generate AAMs from naïve monocytes. Much of this variation appeared to result from differential ability to generate CD68+ macrophages, as most CD68+ cells were also CD163+. Differences in tumor cell potential to generate macrophages was not due to a MCSF-dependent mechanism, nor variance in established pro-AAM factors. TGFα was implicated as a potential signaling molecule produced by tumor cells that could induce macrophage differentiation, which was validated using a CRISPR knockout of TGFA in the OVCAR5 cell line.ConclusionsHGSOC production of TGFα drives monocytes to differentiate into macrophages, representing a central arm of the mechanism by which AAMs are generated in the tumor microenvironment.

Highlights

Alternatively-activated macrophages (AAMs), an anti-inflammatory macrophage subpopulation, have been implicated in the progression of high grade serous ovarian carcinoma (HGSOC)
HGSOC cell lines promote primary monocyte differentiation and polarization towards AAM phenotype We co-cultured primary human monocytes from three unique donors with a panel of five HGSOC cell lines (OV90, OVCAR4, OVCAR3, OVCA432, and OVCAR5) that have been categorized as genomically-consistent with patient samples [32]
Published classical and alternative activation protocols resulted in robust production of CD68+ macrophages, with the classical activation resulting in CD163- cells and the alternative activation resulting in CD163+ for all three monocyte donors (Supplemental Figure 2)

Summary

Introduction

Alternatively-activated macrophages (AAMs), an anti-inflammatory macrophage subpopulation, have been implicated in the progression of high grade serous ovarian carcinoma (HGSOC). Macrophages are vital members of the innate immune system, playing roles in both initiation and resolution of the inflammatory response [6]. This duality of function is primarily due to the plasticity of macrophages, which exist along a spectrum ranging from classically-activated macrophages (CAMs, pro-inflammatory) to alternativelyactivated macrophages (AAMs, anti-inflammatory). Both CAMs and AAMs are derived from monocytes, which infiltrate wound beds or tumor microenvironments in response to secretion of chemokine ligand 2 (CCL2, known as MCP-1). Once monocytes differentiate to macrophages, they continuously interpret microenvironmental cues and respond by adjusting their polarization, altering their position along the inflammatory spectrum [7,8,9]

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